U.S. patent number 4,758,428 [Application Number 06/755,265] was granted by the patent office on 1988-07-19 for multiclass hybrid interferons.
This patent grant is currently assigned to Cetus Corporation. Invention is credited to Abla A. Creasey, David F. Mark.
United States Patent |
4,758,428 |
Mark , et al. |
July 19, 1988 |
Multiclass hybrid interferons
Abstract
New multiclass hybrid interferon polypeptides, their
corresponding encoding recombinant DNA molecules and transformed
hosts which produce the new interferons are described. The amino
acid sequences of these hybrids include at least two different
subsequences, one of which has substantial homology with a portion
of a first class of interferon (e.g., HuIFN-.alpha.) and the other
which has substantial homology with a portion of a second class of
interferon (e.g., HuIFN-.beta.). Data indicates the interferon
activity of .alpha.-.beta. hybrids may be substantially restricted
to either cell growth regulatory activity or antiviral
activity.
Inventors: |
Mark; David F. (Hercules,
CA), Creasey; Abla A. (San Mateo, CA) |
Assignee: |
Cetus Corporation (Emeryville,
CA)
|
Family
ID: |
23334917 |
Appl.
No.: |
06/755,265 |
Filed: |
July 15, 1985 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
463574 |
Feb 3, 1983 |
4569908 |
Feb 11, 1986 |
|
|
340782 |
Jan 19, 1982 |
|
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Foreign Application Priority Data
Current U.S.
Class: |
424/85.4;
435/69.51; 435/811; 530/351; 930/142 |
Current CPC
Class: |
C07K
14/555 (20130101); C07K 14/565 (20130101); A61K
38/00 (20130101); Y10S 930/142 (20130101); Y10S
435/811 (20130101) |
Current International
Class: |
C07K
14/435 (20060101); C07K 14/565 (20060101); C07K
14/555 (20060101); A61K 38/00 (20060101); A61K
045/02 (); C07K 013/00 (); C07K 015/26 (); C12P
021/00 () |
Field of
Search: |
;424/85 ;530/351
;435/68,172.3,811 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Shepard et al., Nature, vol. 294, pp. 563-565, 1981..
|
Primary Examiner: Hazel; Blondel
Attorney, Agent or Firm: Hasak; Janet E. Halluin; Albert
P.
Parent Case Text
This application is a divisional application of U.S. application
Ser. No. 463,574 filed Feb. 3, 1983, now issued as U.S. Pat. No.
4,569,908 on Feb 11, 1986, which is a continuation-in-part
application of U.S. application Ser. No. 340,782, filed Jan. 19,
1982, now abandoned.
Claims
We claim:
1. A multiclass hybrid interferon polypeptide having an amino acid
sequence composed of two distinct amino acid subsequences one of
which subsequences comprises the amino acid sequence 1-73 of the
amino terminal enc of HuIFN-.alpha.1, and the other of which
subsequences comprises the amino acid sequence 74-166 of the
carboxy terminal end of HuIFN-.beta.1.
2. A multiclass hybrid interferon polypeptide having an amino acid
sequence composed of two distinct amino acid subsequences one of
which subsequences comprises the amino acid sequence 1-41 of the
amino terminal enc of HuIFN-.alpha.61A, and the other of which
subsequences comprises the amino acid sequence 47-166 of the
carboxy terminal end of HuIFN-.beta.1.
3. A multiclass hybrid interferon polypeptide having an amino acid
sequence composed of two distinct amino acid subsequences one of
which subsequences comprises the amino acid sequence 1-73 of the
amino terminal eno of HuIFN-.beta.1, and the other of which
subsequences comprises the amino acid sequence 74-167 of the
carboxy terminal end of HuIFN-.alpha.1.
4. A pharmaceutical composition comprising a cell growth regulating
or viral inhibiting effective amount of the multiclass hybrid
interferon polypeptide of claim 1 admixed with a pharmaceutically
acceptable vehicle or carrier.
5. A pharmaceutical composition comprising a cell growth regulating
or viral inhibiting effective amount of the multiclass hybrid
interferon polypeptide of claim 2 admixed with a pharmaceutically
acceptable vehicle or carrier.
6. A pharmaceutical composition comprising a cell growth regulating
or viral inhibiting effective amount of the multiclass hybrid
interferon polypeptide of claim 3 admixed with a pharmaceutically
acceptable vehicle or carrier.
7. A method of regulating cell growth in an animal patient
comprising administering to said patient a cell growth regulating
amount of the multiclass hybrid interferon polypeptide of claim
1.
8. A method of regulating cell growth in a human or other animal
patient comprising administering to said patient a cell growth
regulating amount of the multiclass hybrid interferon polypeptide
of claim 3.
9. A method of treating an animal patient for a viral disease
comprising administering to the patient a viral disease inhibiting
amount of the multiclass hybrid interferon polypeptide of claim 2.
Description
DESCRIPTION
1. Technical Field
This invention is in the field of biotechnology. More particularly
it relates to multiclass hybrid interferon polypeptides,
recombinant DNA that codes for the polypeptides, recombinant
vectors that include the DNA, host organisms transformed with the
recombinant vectors that produce the polypeptides, methods for
producing the hybrid interferon polypeptides, pharmaceutical
compositions containing the polypeptides, and therapeutic methods
employing the polypeptides.
2. Background Art
Since the discovery by Isaacs and Lindenmann of interferon in 1957,
many investigations have been conducted on the efficacy of
interferon for treating various human diseases. Interferon is now
generally thought to have three major clinically advantageous
activities normally associated with it, namely, antiviral activity
(Lebleu et al, PNAS USA, 73:3107-3111 (1976)), cell (including
tumor) growth regulatory activity (Gresser et al, Nature,
251:543-545 (1974)), and immune regulatory activity (Johnson, Texas
Reports Biol Med, 35:357-369 (1977)).
Interferons are produced by most vertebrates in the presence of
certain inducers including viruses. Human interferons (HuIFN) thus
far discovered have been divided into three classes: .alpha.,
.beta., and .gamma.. HuIFN-.alpha. is produced in human leukocyte
cells or in transformed leukocyte cell lines known as
lymphoblastoid lines. HuIFN-.alpha. has been purified to
homogeneity (M. Rubenstein et al, "Human Leukocyte Interferon:
Production, Purification to Homogeneity and Initial
Characterization", PNAS, 76:640-44 (1979)). The pure product is
heterogeneous in size and the various molecular species seem to
have differences in crossspecies antiviral activities (L.S. Lin et
al "Characterization of the Heterogeneous Molecules of Human
Interferons: Differences in cross-species antiviral activities of
various molecular populations in human leukocyte interferons", J
Gen Virol 39:125-130 (1978)). The heterogeneity of the leukocyte
interferon has subsequently been confirmed by the molecular cloning
of a family of closely related HuIFN-.alpha. genes from human
leukocyte cells and from lymphoblastoid cell lines (S. Nagata et
al, "The structure of one of the eight or more distinct chromosomal
genes for human interferon-.alpha.", Nature, 287:401-408 (1980);
D.V. Goeddel et al, "The structure of eight distinct cloned human
leukocyte interferon cDNAs", Nature, 290:20-26 (1981)). However, a
comparison of the DNA and amino acid sequences of the HuIFN-.alpha.
interferons also reveals that many of the sequences exhibit
homology at the nucleotide level, some in the order of 70 percent,
and that the related gene products of these homologous DNA
sequences are also homologous. (D. V. Goeddel et al, "The structure
of eight distinct cloned human leukocyte interferon cDNAs", Nature,
290:20-26 (1981); N. Mantein et al, "The nucleotide sequence of a
cloned human leukocyte interferon cDNA", Gene, 10:1-10 (1980); M.
Streuli et al, "At least three human type .alpha. interferons:
Structure of .alpha.-2", Science, 209:1343-1347 (1980)).
HuIFN-.beta. is produced in human fibroblast cells. Although there
is evidence that human fibroblast cells may be producing more than
one HuIFN-.beta. (P. B. Sehgal and A. D. Sagar, "Heterogeneity of
Poly(I) and Poly(C) induced human fibroblast interferon mRNA
species", Nature, 288:95-97 (1980)), only one species of
HuIFN-.beta. has been purified to homogeneity (E. Knight, Jr.,
"Interferon: Purification and initial characterization from human
diploid cells", PNAS, 73:520-523 (1976); W. Berthold et al,
"Purification and in vitro labeling of interferon from a human
fibroblast cell line", J Biol Chem, 253:5206-5212 (1978)). The
amino terminal sequence of this purified HuIFN-.beta. has been
determined (E. Knight, Jr. et al, "Human fibroblast interferon:
Amino acid analysis and amino terminal amino acid sequence",
Science, 207:525-526 (1981)). Molecular cloning by recombinant DNA
techniques of the gene coding for this interferon has been reported
(T. Taniguchi et al, "Construction and Identification of a
Bacterial Plasmid Containing the Human Fibroblast Interferon Gene
Sequence", Proc Japan Acad, 55 Ser B, 464-469 (1979)). This well
characterized human fibroblast interferon will be referred to as
HuIFN-.beta.1 in the rest of this specification.
Although interferons were initially identified by their antiviral
effects (A. Isaacs and J. Lindenmann, "Virus Interference I. The
Interferon", Proc Royal Soc, Ser B, 147:258-267 (1957)), the growth
regulatory effect of interferons is another biological activity
that has also been well documented (I. Gressor and M. G. Tovey,
"Antitumor effects of interferon" Biochim Biophys Acta, 516:213-247
(1978); W. E. Stewart, "The Interferon System" Springer-Verlag, New
York, 292-304 (1979); A. A. Creasey et al, "Role of G0-G1 Arrest in
the Inhibition of Tumor Cell Growth by Interferon", PNAS,
77:1471-1475 (1980)). In addition, interferon plays a role in the
regulation of the immune response (H. M. Johnsons, Texas Reports on
Biology and Medicine, 35:357-369 (1977)), showing both
immunopotentiating and immunosuppressive effects. Interferon may
mediate the cellular immune response by stimulating "natural
killer" cells in the spontaneous lymphocyte - mediated cytotoxicity
(J. Y. Djeu et al, "Augmentation of mouse natural killer cell
activity by interferon and interferon inducers", J Immun, 122:
175-181 (1979)).
Studies concerning the biological activities of interferons have
been conducted by taking advantage of nucleotide and amino acid
sequence homologies between HuIFN-.alpha.1 and HuIFN-.alpha.2.
Hybrids of the two genes were constructed in vitro by recombinant
DNA techniques such that the DNA sequence coding for the amino
terminus of one gene was fused to the DNA sequence coding for the
carboxy terminus of the other gene (M. Streuli et al, "Target cell
specificity of two species of human interferon-.alpha. produced in
Escherichia coli and of hybrid molecules derived from them", PNAS
78:2848-2852 (1981); P. K. Weck et al, "Antiviral activities of
hybrids of two major human leukocyte interferons", Nucleic Acids
Res, 9:6153-6166 (1981)).
HuIFN-.alpha.1 has a lower specific activity on human WISH cells
than on bovine MDBK cells while HuIFN-.alpha.2 behaves in the
opposite manner. Also, HuIFN-.alpha.1 has some activity on mouse L
cells while HuIFN-.alpha.2 has little activity on mouse cells.
However, the HuIFN-.alpha.2-.alpha.1 hybrid (amino terminal
sequence of HuIFN-.alpha.2 fused to the carboxy terminal sequence
of HuIFN-.alpha.1) has much higher activity on mouse L cells than
on human cells (M. Streuli et al, "Target cell specificity of two
species of human interferon-.alpha. produced in E.coli and of
hybrid molecules derived from them", PNAS, 78:2848-2852 (1981); N.
Stebbing et al, "Comparison of the biological properties of natural
and recombinant DNA derived human interferons", The Biology of the
Interferon System, Elsevier/North-Holland, 25-33 (1981); P. K. Weck
et al, "Antiviral activities of hybrids of two major leukocyte
interferons", Nucleic Acids Res, 9:6153-6166 (1981)). Therefore,
target cell specifications can be altered by making hybrid
proteins.
Although these .alpha.-.alpha. hybrids exhibited changes in target
cell specificity as compared to the parent, it was not demonstrated
that there was any attenuation or any restriction of any of the
three interferon activities.
Under some circumstances, the plural biological activity of
interferon may be undesirable. For example, in the clinical
treatment of patients who have received organ transplants and whose
immune system has been suppressed because of anti-rejection drugs,
administration of interferon to combat viral infection could result
in undesirable stimulation of the immune response system and
consequent rejection of the transplanted organs. Moreover, in
clinical applications it is generally desirable in principle to
focus drug therapy on a particular problem such as viral infection
or tumor growth without the possibility of complicating factors
resulting from other activities of the administered drug. In such
treatment and applications it would be desirable to be able to use
an interferon whose activity is limited to the desired activity.
The present invention provides a novel group of hybrid interferons
that have restricted interferon activity as well as changes in
target cell specificity.
DISCLOSURE OF THE INVENTION
One aspect of the invention is a multiclass hybrid interferon
polypeptide having an amino acid sequence composed of at least two
distinct amino acid subsequences one of which subsequences
corresponds substantially in amino acid identity, sequence and
number to a portion of a first interferon and the other of which
corresponds in amino acid identity, sequence and number to a
portion of a second interferon of a different interferon class from
the first interferon.
A second aspect of the invention is DNA units or fragments
comprising nucleotide sequences that upon expression encode for the
above described multiclass hybrid interferons.
A third aspect of the invention is cloning vehicles (vectors) that
include the above described DNA.
A fourth aspect of the invention is host organisms or cells
transformed with the above described cloning vehicles that produce
the above described multiclass hybrid interferons.
A fifth aspect of the invention is processes for producing the
above described multiclass hybrid interferons comprising
cultivating said transformed host organisms or cells and collecting
the multiclass hybrid interferons from the resulting cultures.
Another aspect of the invention is pharmaceutical compositions
comprising an effective amount of one or more of the above
described multiclass hybrid interferons admixed with a
pharmaceutically acceptable carrier.
Another aspect of the invention is a method of regulating cell
growth in an animal patient comprising administering to said
patient a cell growth regulating amount of one or more of the above
described multiclass hybrid interferons having interferon activity
substantially restricted to cell growth regulatory activity.
Still another aspect of the invention is a method of treating an
animal patient for a viral disease comprising administering to said
patient a viral disease inhibiting amount of one or more of the
above described multiclass hybrid interferons having interferon
activity substantially restricted to antiviral activity.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the amino acid sequence for several different
interferons indicated as .beta.1, .alpha.A through .alpha.H and
.alpha.61A with regions of sequence homology being enclosed by dark
lines. The one letter abbreviations recommended by the IUPAC-IUB
Commission on Biochemical Nomenclature are used; A, alanine; C,
cysteine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G,
glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M,
methionine; N, asparagine; P, proline; Q, glutamine; R, arginine;
S, serine; T, threonine; V, valine; W, tryptophan; and Y,
tyrosine.
FIG. 2 illustrates the structure of plasmid pGW5 used in the
methodology of the invention.
FIG. 3 illustrates the nucleotide sequence between the HindIII site
and the EcoRI site of pGW5, as well as the amino acid sequence of
HuIFN-.alpha.1 which the plasmid expresses.
FIG. 4 illustrates the structure of a plasmid pDM101/trp/.beta.1
used in the methodology of the invention.
FIG. 5 illustrates the nucleotide sequence between the HindIII site
and the BglII sites of the plasmid pDM101/trp/.beta.1 as well as
the amino acid sequence of the expressed HuIFN-.beta.1.
FIG. 6 illustrates the amino acid sequences of HuIFN-.alpha.1 and
HuIFN-.beta.1 at around amino acid 70 of both proteins.
FIG. 7 illustrates the 217 base pair (bp) HindIII-HinfI fragment
and the 285 bp HinfI-BglIII fragment of the HuIFN-.beta.1 gene, as
generated in the methodology of the invention.
FIG. 8 illustrates the 213 base pair HindIII-HinfI fragment and the
65 base pair HinfI-PvuII fragment of the HuIFN-.alpha.1 gene, as
generated in the methodology of the invention.
FIG. 9 illustrates the structure of the plasmid coding for the
hybrid protein of Example I infra.
FIG. 10 is the structure of the coding region of the hybrid gene
incorporated in the plasmid of FIG. 9.
FIG. 11 illustrates the nucleotide sequence of the region coding
for the hybrid protein of Example I, as well as showing the amino
acid sequence of the hybrid protein.
FIG. 12 illustrates the structure of the plasmid coding for the
hybrid protein of Example II, infra.
FIG. 13 illustrates the structure of the coding region of the
hybrid gene incorporated in the plasmid of FIG. 12.
FIG. 14 illustrates the nucleotide sequence of the hybrid gene
shown in FIG. 13, as well as showing the corresponding amino acid
sequence of the hybrid protein expressed by said gene.
FIG. 15 illustrates the structure of plasmid mid p.alpha.61A used
in the methodology of the invention.
FIG. 16 illustrates the nucleotide sequence of the E.coli trp
promoter as well as the nucleotide sequence of the HuIFN-.alpha.61A
gene including some of the flanking 3' non coding region of the
gene which was inserted between the EcoRI and HindIII sites of the
plasmid pBW11. The region coding for the HuIFN-.alpha.61A gene
begins with the ATG codon at position 113 and terminates with the
TGA codon at position 614. The corresponding amino acid sequence of
the HuIFN-.alpha.61A protein is also shown.
FIG. 17 illustrates the nucleotide and amino acid sequences of
HuIFN-.beta.1 and HuIFN-.alpha.61A at around amino acid 40 of both
proteins.
FIG. 18 illustrates the 387 bp EcoRI-PvuII fragment and the 120 bp
(Alpha) HindIII-DdeI fragment of the HuIFN-.alpha.61 gene, as
generated in the methodology of the invention.
FIG. 19 illustrates the 381 bp (Beta) DdeI-BglII fragment of the
HuIFN-.beta.1 gene, as generated in the methodology of the
invention.
FIG. 20 illustrates the structure of a plasmid ptrp3 used in the
methodology of the invention.
FIG. 21 illustrates the structure of the plasmid coding for the
hybrid protein of Example III infra.
FIG. 22 is the structure of the coding region of the hybrid gene
incorporated in the plasmid of FIG. 21.
FIG. 23 illustrates the nucleotide sequence of the region coding
for the hybrid protein of Example III, as well as showing the amino
acid sequence of the hybrid protein.
FIG. 24 depicts a protein gel showing the phosphorylation of the
protein kinase in bovine cells.
MODES FOR CARRYINGOUT THE INVENTION
The hybrid interferons of the invention have an amino acid sequence
composed of at least two distinct amino acid subsequences that are
respectively substantially identical to portions of interferons
from different classes. The term "substantially identical" means
that a subsequence of the hybrid exhibits at least about 70%,
preferably at least about 95%, and most preferably 100% homology
with an amino acid subsequence of a given interferon. Lack of
complete homology may be attributable to single or multiple base
substitutions, deletions, insertions, and site specific mutations
in the DNA which on expression code for the hybrid or given
interferon amino acid sequences. When the hybrid is composed of
more than two subsequences, the additional subsequence(s) may
correspond to other portions of the interferons involved in the
initial two subsequences (e.g., if the initial two sequences are
.alpha.1 and .beta.1, the other sequences are .alpha.1 or .beta.1
or correspond to portions of interferons different from those
involved in the initial two subsequences. Hybrids composed of
.alpha. interferon and .beta. interferon subsequences are
preferred. Hybrids composed only two subsequences (.alpha. and
.beta. ) are particularly preferred. Individual subsequences will
usually be at least about 10 amino acid residues in length, more
usually at least about 30 amino acid residues in length.
Multiclass hybrid interferons of the invention exhibit activity
that is different from the interferon activity exhibited by the
parent interferons of which they are composed. The difference is
manifested as a substantial reduction (relative to the parent
interferons) or elimination of one or two of the three conventional
interferon activities. Preferred hybrids are those whose interferon
activity is substantially restricted to one of the three
activities. Based on data developed to date the interferon activity
of the .alpha.-.beta. interferons appears to be substantially
restricted to either cell growth regulatory or antiviral activity.
In some instances the hybrid interferons also have a host range
(target) cell specificity different from that of the parent
interferons from which they are derived. In other words hybrid
interferons of the invention may exhibit a particular interferon
activity in the cells of one but not another animal species in
which the parent interferons also exhibit activity.
The structural homologies between different classes of interferons
(FIG. 1) permit construction of hybrid DNA molecules coding for the
multiclass human hybrid interferon polypeptides. To construct the
hybrid gene, it is preferred, although not required, that the gene
donating the amino terminal end sequence be fused to some suitable
promoter which directs expression of the gene and contains the
appropriate promoter, operator and ribosomal binding sequence. The
hybrids may be made by selecting suitable common restriction sites
within the respective full genes for the different classes of human
interferon. As an alternative, different restriction sites may be
used for cleavage, followed by repair to blunt ends, followed by
blunt end ligation. In either case, the proper reading frame must
be preserved. Once the desired segments are ligated together, they
are placed in a suitable cloning vector, which is used to transform
suitable host organisms or cells. Where the amino terminal fragment
carries the promoter, operator and ribosomal binding sequence,
expression and biological activity of the resultant hybrids may be
directly assayed. Fusions can be directed to different parts of the
gene by choosing appropriate restriction enzyme sites.
The following examples further illustrate the invention and are not
intended to limit the scope of the invention in any way.
EXAMPLE I
Construction of HuIFN-.alpha.1 .beta.1 Hybrid 1
This example describes the construction of a hybrid interferon,
containing sequences from HuIFN-.alpha.1 and HuIFN-.beta.1. It
involves fusing the amino-terminal end coding region of the
HuIFN-.alpha.1 DNA to the DNA coding for the carboxy-terminal end
region of HuIFN-.beta.1 in such a way that the translational
reading frame of the two proteins are preserved and the resulting
protein being expressed from this hybrid gene will have the amino
acid sequence of HuIFN-.alpha.1 at its amino terminal portion and
the amino acid sequence of HuIFN-.beta.1 at its carboxy terminal
portion.
Purification and Isolation of HuIFN-.alpha.1 and HuIFN-.beta.1 DNA
sequences
The plasmids used in the construction of the HuIFN-.alpha.1.beta.1
Hybrid 1 are plasmids pGW5 and pDM101/trp/.beta.1 containing the
genes coding for HuIFN-.alpha.1 and HuIFN-.beta.1 respectively. The
structure of plasmid pGW5 is shown in FIG. 2 and that of plasmid
pDM101/trp/.beta.1 in FIG. 4.
The plasmid pGW5 was constructed from the plasmid pBR322 by
substituting the region between the EcoRI site to the PvuII site
with the E.coli trp promoter and the DNA sequence coding for the
mature protein of HuIFN-.alpha.1 (FIG. 2). The DNA sequence between
the HindIII site and EcoRI site of pGW5, encoding the mature
protein of HuIFN-.alpha.1, is shown in FIG. 3. Also shown in FIG. 3
is the amino acid sequence of HuIFN-.alpha.1 (IFN-.alpha.D in FIG.
1). The plasmid pGW5 expressed HuIFN-.alpha.1 at high levels in
E.coli. When grown in shake-flasks, about 2.times.10.sup.6 units of
antiviral activity per ml of bacterial culture per A600 can be
detected.
The plasmid pDM101/trp/.beta.1 is a derivative of pBR322 with the
E.coli trp promoter located between the EcoRI and HindIII sites
(FIG. 4). The DNA sequences between the HindIII and BglII sites
encode the mature HuIFN-.beta.1 protein sequence. The nucleotide
sequence together with the amino acid sequence is shown in FIG. 5.
When grown in shake-flasks, the E.coli strain carrying
pDM101/trp/.beta.1 expresses HuIFN-.beta.1 at a level of 10.sup.6
units of antiviral activity per ml of bacterial culture per
A600.
The hybrid gene was constructed by taking advantage of the
homologies between the HuIFN-.alpha.1 gene and the HuIFN-.beta.1
gene at around amino acid 70 of both proteins (FIG. 6). There is a
HinfI restriction site (GATTC) present within this region of both
genes. If both DNA sequences are digested with the enzyme HinfI and
the DNA sequence 5'-proximal to the cutting site of the
HuIFN-.alpha.1 DNA (the arrow in FIG. 6 depicts the cutting site)
is ligated to the DNA sequence 3'-proximal to the cutting site of
HuIFN-.beta.1, a fusion of the two genes is created while
preserving the translational reading frame of both genes.
Since there are several HinfI sites in the coding regions of both
HuIFN-.alpha.1 and HuIFN-.beta.1, it is not possible to carry out a
straightforward exchange of DNA sequences. In the case of
HuIFN-.beta.1, a 502 bp HindIII-BglII fragment containing the whole
coding region from pDM101/trp/.beta.1 is first isolated. The
plasmid DNA was digested with restriction enzymes HindIII and BglII
(R. W. Davis et al, "Advanced Bacterial Genetics", Cold Spring
Harbor Laboratory, pp. 227-230, 1980). (This reference will be
referred to as "Advanced Bacterial Genetics" hereinafter), the DNA
fragments were separated on a 1.5% agarose gel in Tris-Borate
buffer ("Advanced Bacterial Genetics" p 148) and the DNA fragments
visualized by staining with ethidium bromide ("Advanced Bacterial
Genetics", pp 153-154). The appropriate DNA fragment, in this case
a 502 bp fragment, is cut out of the gel, placed in a dialysis
tubing with a minimum amount of 0.1X Tris-Acetate buffer ("Advanced
Bacterial Genetics", p 148) and covered with the same buffer in an
electroelution box and a voltage of 150-200 volts applied for 1
hour. The DNA is then recovered from the buffer in the dialysis
tubing and concentrated by ethanol precipitation. The 502 bp
HindIII-BglII fragment was then digested partially with HinfI to
obtain the 285 bp partial HinfI fragment (denoted as .beta.-B)
coding for the carboxy terminal end of HuIFN-.beta.1 (FIG. 7). The
partial digestion of the DNA fragment was accomplished by using
one-tenth the amount of restriction enzyme required for complete
digestion of the DNA ("Advanced Bacterial Genetics", p 227). The
mixture was incubated at the appropriate temperature for the enzyme
and aliquots of the digestion mixture were removed at 10-minute
intervals for up to 1 hour. The aliquots were then loaded onto a
gel and the DNA fragments analyzed. The time point that provides
the highest yield of the DNA fragment needed is chosen for a
preparative digestion with the restriction enzyme and the
appropriate fragment purified from the gel by electroelution. The
other HindIII-BglII fragment, (.beta.-C in FIG. 9) consisting of
the plasmid pDM101 and trp promoter, is also saved and used in the
vector for the HuIFN-.alpha.1.beta.1 hybrid.
In the case of HuIFN-.alpha.1, pGW5 is digested with HindIII and
PvuII and a 278 bp fragment which contains two HinfI sites is
purified from the digest. This fragment is then digested partially
with HinfI to obtain two fragments, a 213 bp HindIII-HinfI fragment
(.alpha.-A) and a 65 bp HinfI-PvuII fragment (.alpha.-B) (FIG.
8).
Vector Preparation and Selection
Assembly of the plasmid for the direct expressions of the
HuIFN-.alpha.1 .beta.1 interferon gene can be constructed by
ligating fragments .alpha.-A, .beta.-B and .beta.-C together as
shown in FIG. 9. The ligated DNA was then used to transform
competent E.coli cells ("Advanced Bacterial Genetics" pp 140-141).
Transformants were plated onto broth plates containing 50 .mu.g per
ml of ampicillin and incubated at 37.degree. C. Ampicillin
resistant colonies were grown up in rich medium in the presence of
50 .mu.g/ml of ampicillin and plasmid DNA isolated from each
individual clone ("Advanced Bacterial Genetics", pp 116-125).
The gene structure of the desired hybrid clone is shown in FIG. 10.
The correct hybrid clone was identified by digesting the plasmid
DNA with the restriction enzymes HindIII and BglII and screening
for the presence of a 498 bp restriction fragment on 1.5% agarose
gel in Tris-Borate buffer ("Advanced Bacterial Genetics", p 148).
To further characterize the hybrid clone, the plasmid DNA was
digested with HinfI and screened for the presence of the 145 bp and
167 bp restriction fragments. By following this scheme, a number of
hybrid clones were identified, one of which (denoted
pDM101/trp/hybrid 41) was selected for further characterization and
culturing to produce the hybrid interferon.
The nucleotide sequence of the region coding for the hybrid protein
is shown in FIG. 11. Also shown in FIG. 11 is the amino acid
sequence of the hybrid protein. This hybrid interferon is denoted
HuIFN-.alpha.1 .beta.1 Hybrid 1 herein. The amino terminal portion
of this polypeptide starting with methionine is composed of the
amino acid sequence 1-73 of HuIFN-.alpha.1 and the carboxy terminal
portion is composed of amino acids 74-166 of HuIFN-.beta.1.
The E.coli strain carrying pDM101/trp/hybrid 41 was grown in
minimal medium containing 50 .mu.g/ml of ampicillin to express the
hybrid protein. The culture was harvested when it reached A600=1.0,
concentrated by centrifugation, resuspended in buffer containing 50
mM Tris-HCL pH 8.0, 10 mM ethylenediaminetetraacetic acid (EDTA),
15% sucrose and 1% sodium dodecylsulfate (SDS), and the cells lysed
by sonication in a Branson Sonicator. The cell free extract was
assayed for (1) inhibiting the growth of transformed cells, (2)
activating natural killer cells, and (3) antiviral activity.
Biological Testing of HuIFN-.alpha.1 .beta.1 Hybrid 1
(1) Growth Inhibition Assays
Bacterial extracts made from the E.coli strain carrying
pDM101/trp/hybrid 41, together with various control extracts, were
assayed for their ability to inhibit the growth of two human tumor
cell lines, the Daudi line (American Type Culture Collection,
Catalog of Cell Strains III, 3rd Edition, Rockville, MD (1979)) and
the melanoma line HS294T Clone 6 (A. A. Creasey et al, PNAS,
77:1471-1475, (1980); A. A. Creasey et al, Exp Cell Res,
134:155-160 (1981)).
(a) Inhibition of Growth of Daudi Cells
About 2.times.10.sup.4 cells are seeded into each well of a sterile
96-well round bottom microtiter plate. Cells are then incubated
overnight at 37.degree. C. Bacterial extracts together with the
appropriate controls are added to the cells and then allowed to
incubate at 37.degree. C. for three days. On the third day, cells
are pulse labeled with 4 .mu.Ci well of .sup.3 H-thymidine for 2-3
hours. The labeling is terminated by addition of 5% trichloroacetic
acid (TCA) to precipitate the nucleic acids. The precipitates are
filtered and the filters are counted in the scintillation counter.
The results for the cells incubated with the bacterial extracts are
compared to the results for the controls to obtain a percent
inhibition of growth. The results are reported in Table I
below.
(b) Inhibition of HS294T Clone 6
About 1.5.times.10.sup.4 cells are seeded into each well of a
sterile, flexible 48-well flat bottom tissue culture plate. Cells
are incubated overnight at 37.degree. C. with 10%
CO.sub.2.Bacterial extracts together with various controls are
added to the cells and then incubated for three days at 37.degree.
C. On the third day, cells are pulse labeled with 2 .mu.Ci/well of
.sup.3 H-thymidine for 2-3 hours. The labeling reactions is
terminated by addition of cold TCA in 0.3% Na.sub.4 P.sub.2 O.sub.7
(TP). Plates are washed two times with TP solution and three times
with cold absolute ethanol, and left to dry at room temperature. A
sheet of adhesive tape is stuck to the bottom of the assay plate,
securing all the wells in place. The plate is then run through a
hot wire cutter. The top of the plate is removed and the individual
wells are picked off the adhesive tape and put into scintillation
vials containing 5 ml of scintillation fluid and counted in the
scintillation counter. Percent growth inhibition was obtained as
above. The results are also reported in Table I below.
TABLE I ______________________________________ Percent Inhibition
of U/ml or Growth Cell Lines *dilution of HS294T HuIFN Extract
Daudi Clone 6 ______________________________________ .alpha..sup.1
100 70 0 500 80 9 .beta..sup.1 100 68 43 500 72 80 Hybrid of
*1:2000 46 4 Example I *1:20,000 24 0
______________________________________ Note: Percent inhibition of
growth by negative control (pDM101/trp) was include in the
calculations to obtain the numbers shown above)
As reported in Table I the hybrid interferon HuIFN-.alpha.1 .beta.1
Hybrid 1 inhibited the growth of Daudi cells but it did not inhibit
the HS294T Clone 6 cells. Since the HS294T Clone 6 cells are
resistant to HuIFN-.alpha.1 the hybrid appears to be behaving like
HuIFN-.alpha.1 in these tests. Therefore, it appears that since the
hybrid has the HuIFN-.alpha.1 amino terminal sequence as its amino
terminus, that portion of the protein may carry the determinant
which governs cell specificity.
(2) Stimulation of Natural Killer Cells
Whole blood is obtained from a donor and kept clot-free by adding
EDTA. Lymphocytes are separated by centrifugation on a
Ficoll/Hypaque gradient. The upper band of lymphocytes is harvested
and washed. Interferon samples and various control samples are
diluted into 1 ml of Dulbecco's Modified Eagle's Medium (DME)
containing 10% fetal calf serum (FCS) and then mixed with 1 ml of
lymphocytes (10.sup.7 cells) and incubated at 37.degree. C. for 18
hours. The treated lymphocytes are then washed and resuspended in
RPMI 1640 medium containing 10% FCS.
Two hours before the lymphocytes are harvested, the target cells
(Daudi line) are labeled with .sup.51 Cr by incubating
2.times.10.sup.6 Daudi cells with 100 .mu.Ci of .sup.51 Cr in 1 ml
of RPMI 1640. After two hours, the target cells are washed four
times to remove excess label, concentrated by centrifugation and
resuspended to 2.times.10.sup.5 cells per ml in RPMI 1640. About
2.times.10.sup.4 labeled target cells are added to each well of a
microtiter plate. Primed lymphocytes together with unprimed
controls are added to the target cells in triplicate and incubated
for four hours at 37.degree. C. The plate is then centrifuged and
100 .mu.l of media is removed from each well and counted in the
gamma counter. Percent killing by the activated natural killer
cells is dependent on the interferon concentration. Thus, small
amounts of interferon will result in a small percentage of killing
and minimal lysis of target cells. By determining the amount of
label released into the medium, the amount of natural killer
activity can be quantitated. The results of the tests are reported
in Table II below.
TABLE II ______________________________________ ACTIVATION OF
NATURAL KILLER CELLS U/ml or *dilution of Percent HuIFN extract
Killing (%) ______________________________________ .alpha..sup.1
100 39 10 29 .beta..sup.1 100 38 10 2 Hybrid of *1:1000 13 Example
I Controls: pDM101/trp/ *1:1000 10 Cell Control 7 (Spontaneous
release of label) ______________________________________
As reported in Table II, the hybrid interferon showed substantially
less natural killer activity than HuIFN-.beta.1 and
HuIFN-.alpha.1.
(3) Antiviral Assays
Interferon antiviral activity in bacterial extracts was determined
by comparison with NIH interferon standards using cytopathic effect
(CPE) inhibition assays as reviewed previously (W. E. Stewart, "The
Interferon System" Springer-Verlag, 17-18, (1979)). The assays were
performed on two different cell lines the human trisomic 21 line
(GM2504), and the bovine MDBK line, with vesicular stomatitis virus
as the challenge virus within the limits of the sensitivity of the
CPE inhibition assay (.gtoreq.30 U/ml) no antiviral activity in the
bacterial extracts containing the hybrid interferon of Example I
was detected.
EXAMPLE II
Construction of HuIFN-.beta.1.alpha.1 Hybrid1
This example describes the construction of a hybrid interferon
containing sequences from HuIFN-.alpha.1 and HuIFN-.beta.1. It
involves the fusion of the amino terminal coding region of the
HuIFN-.beta.1 DNA to the DNA coding for the carboxy terminal region
of HuIFN-.alpha.1 in such a way that the translational reading
frame of the two genes are preserved and the resulting protein
being expressed from this hybrid gene will have the amino acid
sequence of HuIFN-.beta.1 at its amino terminus and the amino acid
sequence of HuIFN-.alpha.1 at its carboxy terminus.
Purification and Isolation of HuIFN-.alpha.1 and HuIFN-.beta.1 DNA
Sequences.
The plasmids used in the construction of HuIFN-.beta.1.alpha.1
hybrid 1 are plasmids pGW5 and pDM101/trp/.beta.1 as set forth in
Example I.
As in Example I, the hybrid gene of this example was constructed by
taking advantage of the homologies between HuIFN-.alpha.1 and
HuIFN-.beta.1 at around amino acid 70 of both proteins (FIG. 6).
The DNA sequence 5'-proximal to the cutting site of the
HuIFN.beta.1 DNA (the arrow in FIG. 6 depicts the cutting site), is
ligated to the DNA sequence 3'-proximal to the cutting site of
HuIFN-.alpha.1, to create a fusion of the two genes while
preserving the translational reading frame of both genes.
Since there are several HinfI sites in the coding regions of both
HuIFN-.alpha.1 and HuIFN-.beta.1 it is not possible to carry out a
straightforward exchange of DNA sequences. Thus the procedures of
Example I were followed for the isolation of the 217 bp fragment
(denoted as .beta.-A) as shown in FIG. 7.
In the case of HuIFN-.alpha.1, pGW5 was digested with HindIII and
PvuII and two fragments were purified. One of the fragments is 278
bp in length (the small fragment) and contains two HinfI sites.
This fragment is digested partially with HinfI to obtain two
fragments, a 213 bp HindIII-HinfI fragment (.alpha.-A) and a 65 bp
HinfI-PvuII fragment (.alpha.-B) (FIG. 8). The other HindIII-PvuII
fragment containing the carboxy terminus coding region of
HuIFN-.alpha.1 (.alpha.-C fragment) is saved for use as vector for
cloning the hybrid.
Vector Preparation and Selection
The hybrid can be constructed by ligating 0 fragments .beta.-A,
.alpha.-B and .alpha.-C together as shown in FIG. 12. This ligated
DNA was then used to transform competent E.coli cells.
Transformants were plated onto broth plates containing 50 .mu.g/ml
of ampicillin and incubated at 37.degree. C. Ampicillin resistant
colonies were grown up in rich medium in the presence of 50
.mu.g/ml of ampicillin and plasmid DNA isolated from each
individual clone.
The gene structure of the desired hybrid clone is shown in FIG. 13.
Therefore, the correct hybrid clone could be identified by
digesting the plasmid DNA with the restriction enzyme PvuII and
screening for the presence of the characteristic 141 bp PvuII
fragment (FIG. 13) on 5% polyacrylamide gel. To further
characterize the hybrid clone, the plasmid DNA was digested with
HinfI and screened for the presence of the 197 bp, 159 bp, 129 bp,
and 39 bp HinfI restriction fragments. By following this scheme, a
number of hybrid clones were identified, one of which (denoted
pDM101/trp/hybrid 1) was selected for further characterization and
culturing to produce the hybrid interferon.
The nucleotide sequence of the region coding for the hybrid protein
is shown in FIG. 14. Also shown in FIG. 14 is the amino acid
sequence of the hybrid protein. This hybrid interferon is denoted
HuIFN-.beta.1.alpha.1 Hybrid 1 herein. The amino terminal portion
of this polypeptide starting with methionine is composed of the
amino acid sequence 1-73 of HuIFN-.beta.1 and the carboxy terminal
portion is composed of amino acids 74-166 of HuIFN-.alpha.1.
Biological Testing of HuIFN-.beta.1.alpha.1 Hybrid 1
The assays used to determine interferon activities were identical
to those used in Example I. The following Tables III and IV report
the results of the cell growth regulatory assays and the natural
killer cell activity assay.
TABLE III ______________________________________ Percent Inhibition
of U/ml or Growth Cell Lines *dilution of HS294T HuIFN Extract
Daudi Clone 6 ______________________________________ .alpha..sup.1
100 70 0 500 80 9 .beta..sup.1 100 68 43 500 72 80 Hybrid of
*1:2000 80 16 Example II *1:20,000 23 28
______________________________________ Note: Percent inhibition of
growth by negative control (pDM101/trp) was include in the
calculations to obtain the numbers shown above.
As reported and in contrast to Example I, the hybrid interferon of
Example II inhibited the growth of both Daudi and HS294T Clone 6
cells, thus behaving like HuIFN-.beta.1. Therefore,
HuIFN-.beta.1.alpha.1 Hybrid 1 supports the hypothesis expressed in
Example I that the amino terminal portion of the interferon carries
the determinat which governs cell specificity.
TABLE IV ______________________________________ ACTIVATION OF
NATURAL KILLER CELLS U/ml or *dilution of Percent HuIFN Extract
Killing (%) ______________________________________ .alpha..sup.1
100 39 10 29 .beta..sup.1 100 38 10 2 Hybrid of *1:000 14 Example
II Controls: pDM101/trp *1:000 10 Cell Control 7 (Spontaneous
release of label) ______________________________________
Antiviral assays were carried out using the HuIFN-.beta.1.alpha.1
Hybrid 1. Within the realm of sensitivity of the CPE inhbition
assay no antiviral activity in the bacterial extracts containing
the hybrid interferon was detected.
EXAMPLE III
Construction of HuIFN-.alpha.61A.beta.1 Hybrid
This example describes the construction of a hybrid interferon
containing sequences from HuIFN-.alpha.61A and HuIFN-.beta.1. It
involves the fusion of the amino acid terminal coding region of the
HuIFN-.alpha.61A DNA to the DNA coding for the carboxy terminal
region of HuIFN-.beta.1 in such a way that the translational
reading frame of the two genes are preserved and the resulting
protein being expressed from this hybrid gene will have the amino
acid sequence of HuIFN-.alpha.61A at its amino terminus and the
amino acid sequence of HuIFN-.beta.1 at its carboxy terminus.
Purification and Isolation of HuIFN-.alpha.61A and HuIFN-.beta.1
DNA Sequences
The plasmids used in the construction of HuIFN-.alpha.61A.beta.1
hybrid are plasmids p.alpha.61A and pDM101/trp/.beta.1 Example I
and FIG. 4).
Preparation of plasmid p.alpha.61A
In order to assemble the plasmid p.alpha.61A, the Namalwa cell
human IFN enriched mRNA was used to construct complementary DNA
(cDNA) clones in E.coli by the G/C tailing method using the PstI
site of the cloning vector pBR322 (Bolivar, F., et al, Gene,
2:95-113 (1977)). A population of transformants containing
approximately 50,000 individual cDNA clones was grown in one liter
of medium overnight and the total plasmid DNA was isolated.
The sequences of two IFN-.alpha. clones (IFN-.alpha.1 and
IFN-.alpha.2) have been published (Streuli, M., et al, Science,
209:1343-1347 (1980)). Examination of the DNA sequences of these
two clones revealed that the restriction enzyme XhoII would excise
a 260 bp fragment from either the IFN-.alpha.1 or the IFN-.alpha.2
gene (see FIG. 1). XhoII was prepared in accordance with the
process described by Gingeras, T. R., and Roberts, R. J., J Mol
Biol, 118:113-122 (1978).
One mg of the purified total plasmid DNA preparation was digested
with XhoII and the DNA fragments were separated on a preparative 6%
polyacrylamide gel. DNA from the region of the gel corresponding to
260 bp was recovered by electroelution and recloned by ligation
into the BamHI site of the single strand bacteriophage m13:mp7.
Thirty-six clones were picked at random, the single stranded DNA
isolated therefrom, and the DNA was sequenced. The DNA sequences of
four of these clones were homologous to known IFN-.alpha. DNA
sequences. Clone mp7:.alpha.-260, with a DNA sequence identical to
IFN-.alpha.1 DNA (Streuli, M. et al, Science, 209:1343-1347 (1980))
was chosen as a highly specific hybridization probe for identifying
additional IFN-.alpha. DNA sequences. This clone is hereinafter
referred to as the "260 probe."
In order to isolate other IFN-.alpha. gene sequences, a .sup.32
P-labelled 260 probe was used to screen a library of human genomic
DNA by in situ hybridization. The human gene bank, prepared by
Lawn, R. M., et al, Cell, 15:1157-1174 (1978), was generated by
partial cleavage of fetal human DNA with HaeIII and AluI and cloned
into bacteriophage .lambda. Charon 4A with synthetic EcoRI linkers.
Approximately 800,000 clones were screened, of which about 160
hybridized with the 260 probe. Each of the 160 clones was further
characterized by restriction enzyme mapping and comparison with the
published restriction maps of 10 chromosomal IFN genes (Nagata, S.,
et al, J Interferon Research, 1:333-336 (1981)). One of the clones,
hybrid phage .lambda.4A:.alpha.61 containing a 18 kb insert, was
characterized as follows. A DNA preparation of .lambda.4A:.alpha.61
was cleaved with HindIII, Bg1II, and EcoRI respectively, the
fragments separated on an agarose gel, transferred to a
nitrocellulose filter (Southern, E. M., J Mol Biol, 98:503-517
(1977)) and hybridized with 32P-labelled 260 probe. This procedure
localized the IFN-.alpha.61 gene to a 1.9 kb Bg1II restriction
fragment which was then isolated and recloned, in both
orientations, by ligation of the fragment into BamHI cleaved
m13:mp7. The two subclones are designated mp7:.alpha.61-1 and
mp7:.alpha.61-2. The -1 designation indicates that the
single-stranded bacteriophage contains insert DNA complementary to
the mRNA (the minus strand) and the -2 designation indicates that
the insert DNA is the same sequence as the mRNA (the plus
strand).
The Sanger dideoxy-technique was used to determine the DNA sequence
of the HuIFN-.alpha.61A gene. The DNA sequence of the
IFN-.alpha.61A gene and the amino acid sequence predicted therefrom
differ substantially from the other known IFN-.alpha. DNA and
IFN-.alpha. amino acid sequences. In this regard Goeddel, D.V., et
al Nature (1981) 290:20-26 discloses the DNA sequence of a partial
IFN cDNA clone, designated LeIF-G. The sequence of the partial
clone is similar to the 3'-end of the IFN-.alpha.61A DNA sequence,
except for a nucleotide change in the codon for amino acid 128. As
compared to the partial clone the IFN-.alpha.61A gene contains
additional DNA that codes for the first 33 amino acids of
IFN-.alpha.61A.
Assembly of the p.alpha.61A plasmid involved replacing the DNA
fragment encoding the 23 amino acid signal polypeptide of
preinterferon with a 120 bp EcoRI/Sau3A promoter fragment E.coli
trp promoter, operator, and trp leader ribosome binding site
preceoperator, ding an ATG initiation codon and using HindIII site
that was inserted, 59 nucleotides 3'- of the TGA translational stop
codon, to insert the gene into the plasmid pBW11 (a derivative of
pBR322 having a deletion between the HindIII and PvuII sites). The
complete DNA sequence of the promoter and gene fragments inserted
between the EcoRI and HindIII sites of pBW11 is shown in FIG. 16
which also shows the exact location of relevant cloning sites.
Details of the construction are described below.
The coding region for mature IFN-.alpha.61 has three Sau3A sites,
one of which is between codons for amino acids 2 and 3. A synthetic
HindIII site was inserted 59 nucleotides 3'- of the coding region
and the resulting construct was subjected to a HindIII/partial
Sau3A digest. A 560 bp fragment was isolated from the digest. This
fragment and a 120 bp EcoRI to Sau3A E.coli promoter fragment were
ligated together in a +three way directed ligation into the EcoRI
to HindIII site of pBW11. The promoter fragment, contained a
synthetic HindIII restriction site, ATG inititation codon, the
initial cysteine codon (TGT) common to all known IFN-.alpha.s, and
a Sau3A "sticky end". The ligation mixture was used to transform
E.coli . The final expression plasmid obtained, p.alpha.61A, is
shown in FIG. 15.
As in Examples I and II, the hybrid gene of the example was
constructed by taking advantage of the homologies between
HuIFN-.alpha.61A (the DNA sequence of the HuIFN-.alpha.61A gene and
the amino acid sequence it encodes are shown in FIG. 16) and
HuIFN-.beta.1 at around amino acid 40 of both proteins (FIG. 17).
The DNA sequence 5'-proximal to the DdeI restriction enzyme cutting
site of the HuIFN-.alpha.61A DNA (the arrow in FIG. 17 depicts the
cutting site), is ligated to the DNA sequence 3'-proximal to the
cutting site of HuIFN-.beta.1, to create a fusion of the two genes
while preserving the translational reading frame of both genes.
Since there are several DdeI sites in the coding regions of both
HuIFN-.alpha.61A and HuIFN-.beta.1, and the DdeI cohesive ends are
not identical, therefore, it is not possible to carry out a
straightforward exchange of DNA fragments. Thus variations of the
procedures described in Examples I and II were used.
In the case of HuIFN-.alpha.61A, p.alpha.61A was digested with
EcoRI and PvuII and the 387 bp fragment containing three DdeI sites
was purified. This fragment was digested partially with DdeI, the
cohesive ends repaired to a blunt end by the action of DNA
Polymerase I Klenow fragment as described by Maniatis et al.,
("Molecular Cloning" Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y. p. 113-114 (1982)). The repaired DNA fragments were
then digested with HindIII and the 120 bp fragment (denoted as
Alpha) purified from an acrylamide gel (FIG. 18).
In the case of HuIFN-.beta.1, pDM101/trp/.beta.1 was digested with
EcoRI and BamHI and the smaller fragment, containing the interferon
gene purified (FIG. 4). This fragment was partially digested with
DdeI, the cohesive ends removed by treatment with S1 nuclease as
described by Maniatis et al., ("Molecular Cloning", Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y. p. 140 and 237-238
(1982)). The S1 nuclease treated DNA was then digested with Bg1II
and the 381 bp fragment (denoted as Beta) purified (FIG. 19).
Vector preparation
The plasmid ptrp3 (FIG. 20) is a derivative of pBR322, with the
EcoRI - ClaI region replaced by the E.coli trp promoter sequence.
This plasmid was digested with HindIII and BamHI and the large
plasmid fragment containing the E.coli trp promoter was purified
(FIG. 20).
The hybrid was constructed by ligating this vector fragment to the
Alpha and Beta fragments as shown in FIG. 21. This ligated DNA was
transformed into competent E.coli cells and plated on plates
containing ampicillin. Resistant colonies were grown up
individually in rich medium and plasmid DNA isolated from them. The
plasmid DNA were digested with DdeI and screened on acrylamide gels
for the presence of the 91 bp and 329 bp DdeI fragments
characteristic of the hybrid as shown in FIG. 22. A number of
hybrid clones were identified, one of which (denoted as
p.alpha..beta.62) was selected for further characterization and
culturing to produce the hybrid interferon.
The nucleotide sequence of the region coding for the hybrid protein
is shown in FIG. 23. Also shown in FIG. 23 is the amino acid
sequence of the hybrid protein. This hybrid interferon is
denoted
25 HuIFN-.alpha.61A.beta.1 herein. The amino terminal portion of
this polypeptide starting with methionine is composed of the amino
acid sequence 1-41 of HuIFN-.alpha.61A and the carboxy terminal
portion is composed of amino acids 47-166 of HuIFN-.beta.1.
Biological Testing of HuIFN-.alpha.61A.beta.l Hybrid
The assays used to determine interferon activities were identical
to those used in Examples I and II. However, an additional assay
was incorporated, the protein kinase phosphorylation assay, to
confirm the change we observed in host range specificity of the
antiviral activity of this hybrid as compared to its parents.
Growth Inhibition and Natural Killer Cell Assays
No inhibition of either Daudi or Clone 6 cells was exhibited.
Similarly no activation of natural killer cells was detected.
Antiviral Assays
We performed our biological antiviral assays as described for
Examples I and II on two different cell lines: the human trisomic
21 cell line (GM2504), and the bovine MDBK line, with vesicular
stomatitis virus as the challenge virus. Our results are summarized
in Table V. As compared to the previous two examples,
HuIFN-.alpha.61A.beta.1 had antiviral activity on bovine cells
(.about.10.sup.3 U/ml), but no detectable antiviral activity on
human GM2504 cells.
69K Protein Phosphorylation
The biological activity of interferons has usually been studied by
infecting treated cell cultures and measuring the inhibition of
virus replication. A more direct approach would be to measure, in
the cells, some interferon-induced biochemical changes associated
with the establishment of the antiviral state. One of the clearest
biochemical alterations observed after interferon treatment is an
impairment of viral protein synthesis (M. Revel,
"Interferon-Induced Translational Regulation," Texas Rep Biol Med
35:212-219 (1977)). Several cellular inhibitions of mRNA
translation have been identified in interferonm-treated cells and
shown, after purification, to be enzymes that act on various
components of the mRNA translation machinery. One cellular enzyme
is a specific protein kinase, phosphorylating a 69,000 Mr
polypeptide (P.sub.1) and the small subunit of eukaryotic
initiation factor 2 (eIF-2). (For review, see C. Samuel,
"Procedures for Measurement of Phosphorylation of Ribosome
Associated Proteins in Interferon Treated Cells." Methods in
Enzymology, 79:168-178. (1981)). Phosphorylation of protein P.sub.1
is considered one of the most sensitive biochemical markers of
interferon action and is significantly enhanced in
interferon-treated cells as compared to untreated cells. To confirm
the change in the host range in the antiviral activity of
HuIFN-.alpha.61A.beta.1, we used the protein kinase phosphorylation
assay as has been described by A. Kimchi et al, "Kinetics of the
Induction of Three Translation-Regulatory Enzymes by Interferon",
Proc Natl Acad Sci, 76:3208-3212 (1979). We have found that the
HuIFN-.alpha.61A.beta.1 indicated in FIG. 24 as .alpha..beta.62,
induced the phosphorylation of the kinase in the bovine MDBK cells
and not in the human GM2504 cells. The + and - symbols in FIG. 24
indicate the presence or absence of polyIC double stranded RNA in
the reaction. The arrow points to the bands indicating the
interferon-induced phosphorylation of the 69K double stranded RNA
dependent cellular protein (P.sub.1). These results confirm the
antiviral activity of HuIFN-.alpha.61A.beta.1 on bovine cells.
TABLE V ______________________________________ Antiviral activity
of recombinant parent and hybrid interferons on bovine and human
cells in culture Cell Line Human Fibroblasts Bovine Fibroblasts
(GM2504) (MDBK) IFN/type IFN Titer (U/ml)
______________________________________ IFN-.alpha.61A .sup.
>10.sup.6 10.sup.6 IFN-.beta.1 5 .times. 10.sup.5 5 .times.
10.sup.3 IFN-.alpha.61A.beta.1 <30 10.sup.3 trp control <30
<30 .sup. ______________________________________
The cell growth regulating activity exhibi
ted by certain .alpha.-.beta. hybrid interferons makes these
hybrids potentially useful for treating tumors and cancers such as
osteogenic sarcoma, multiple myeloma, Hodgkin's disease, nodular,
poorly differentiated lymphoma, acute lymphocytic leukemia, breast
carcinoma, melanoma, and nasopharyngeal carcinoma. Because of their
restricted activity such treatment is not expected to be associated
with side effects such as immunosuppression that often is observed
with conventional nonhybrid interferon therapy. Also it is -
expected that the .alpha.-.beta. hybrid interferons exhibiting
interferon activity restricted to antiviral activity may be used to
treat viral infections with a potential for interferon therapy such
as encephalomyocarditis virus infection, influenza and other
respiratory tract virus infections, rabies and other viral zoonoses
and arbovirus infections.
Pharmaceutical compositions that contain a hybrid interferon as an
active ingredient will normally be formulated with an appropriate
solid or liquid carrier depending upon the particular mode of
administration being used. For instance, parenteral formulations
are usually injectable fluids that use pharmaceutically and
physiologically acceptable fluids such as physiological saline,
balanced salt solutions, or the like as a vehicle. Oral
formulations, on the other hand, may be solid, eg tablet or
capsule, or liquid solutions or suspensions. The hybrid interferon
will usually be formulated as a unit dosage form that contains
approximately 100 .mu.g of protein per dose.
The hybrid interferons of the invention may be administered to
humans or other animals on whose cells they are effective in
various manners such as orally, intravenously, intramuscularly,
intraperitoneally, intranasally, intradermally, and subcutaneously.
The particular mode of administration and dosage regimen will be
selected by the attending physician taking into account the
particulars of the patient, the disease and the disease state
involved. For instance, viral infections are usually treated by
daily or twice daily doses over a few days to a few weeks; whereas
tumor or cancer treatment typically involves daily or multidaily
doses over months or years. The same dose levels as are used in
conventional nonhybrid interferon therapy may be used. A hybrid
interferon may be combined with other treatments and may be
combined with or used in association with other chemotherapeutic or
chemopreventive agents for providing therapy against neoplasms or
other conditions against which it is effective.
Modifications of the above described modes for carrying out the
invention, such as, without limitation, use of alternative vectors,
alternative expression control systems in the vector, and
alternative host microorganisms and other therapeutic or related
uses of the hybrid interferons, that are obvious to those of
ordinary skill in the biotechnology, pharmaceutical, medical and/or
related fields are intended to be within the scope of the following
claims.
* * * * *